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Methods. 2016 Jul 1;103:68-76. doi: 10.1016/j.ymeth.2016.02.003. Epub 2016 Feb 4.

Detection and quantification of RNA 2'-O-methylation and pseudouridylation.

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Department of Process Development, Bristol-Myers Squibb Company, 6000 Thompson Road, East Syracuse, NY 13027, USA. Electronic address:
Department of Plant and Microbial Biology, University of California, 565 Li Ka Shing Center #3370, Berkeley, CA 94720-337, USA.
Department of Biochemistry and Biophysics, University of Rochester Medical Center, School of Medicine and Dentistry, 601 Elmwood Avenue, Box 712, Rochester, NY 14642, USA. Electronic address:


RNA-guided RNA modification is a naturally occurring process that introduces 2'-O-methylation and pseudouridylation into rRNA, spliceosomal snRNA and several other types of RNA. The Box C/D ribonucleoproteins (RNP) and Box H/ACA RNP, each containing one unique guide RNA (Box C/D RNA or Box H/ACA RNA) and a set of core proteins, are responsible for 2'-O-methylation and pseudouridylation respectively. Box C/D RNA and Box H/ACA RNA provide the modification specificity through base pairing with their RNA substrate. These post-transcriptional modifications could profoundly alter the properties and functions of substrate RNAs. Thus it is desirable to establish reliable and standardized modification methods to study biological functions of modified nucleotides in RNAs. Here, we present several sensitive and efficient methods and protocols for detecting and quantifying post-transcriptional 2'-O-methylation and pseudouridylation.


2′-O-methylation; Box C/D guide RNA; Box H/ACA guide RNA; Post-transcriptional modification; Pseudouridylation; RNA

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